POS0617 THE GASOTRANSMITTER HYDROGEN SULFIDE PROTECTS AGAINST CALCIFIC TENDINOPATHY VIA INHIBITION OF LYSYL OXIDASES
Authors: S. Nasi, I. Bernabei, E. Faure, D. Kronenberg, J. Bertrand, D. Ehirchiou, R. Stange, T. Hügle, N. Busso
Journal: Annals of the Rheumatic Diseases (2023)

<h3>Background</h3> Pathological calcification ((PC), <i>i.e.</i> the deposition of calcium-containing crystals) in tendons is a hallmark of calcific tendinopathy (CT), a disease associated with pain, tendon rupture, and disability. Currently, only symptomatic treatments exist for CT and none of them target specifically calcification. Intriguingly, the gasotransmitter H<sub>2</sub>S has recently emerged as a potential anti-calcifying molecule [1]. <h3>Objectives</h3> To establish in CT the potential anti-calcifying effect of hydrogen sulfide (H<sub>2</sub>S) and of the H<sub>2</sub>S-producing enzyme cystathionine gamma lyase (CSE), and the underlying mechanisms involved. <h3>Methods</h3> Wild type (WT) and CSE knock-out (KO) tenocytes were isolated from adult mice Achilles' tendons. Tenocyte were cultured in αMEM + 10% FBS + 50μg/ml ascorbic acid (Control medium) in presence of 10% calciprotein particles (calcification medium, CM). Spontaneous Achilles' tendon CT in old mice (35 weeks old) and surgery-induced Achilles' tendon CT in8-12 weeks old mice were quantified by micro computed tomography. Dynamic Young's modulus was measured in 35 weeks old mice as previously described [2]. Immunohistochemistry was performed on human and murine tendon sections with anti-CSE and anti-LOX and anti-LOXL2rabbit polyclonal antibodies. LOX expression was analyzed by qPCR and by Western-blot. LOX(L) activity was quantified in cells supernatants and lysates. <h3>Results</h3> <i>In vitro</i>, tenocyte calcification (in CM) was inhibited by exogenous H<sub>2</sub>S-donors, while it was exacerbated in CSE KO tenocytes, producing as expected less H<sub>2</sub>S than WT. Reduced calcification in tenocytes exposed to H<sub>2</sub>S was accompanied by decreased expression of genes coding for BMP2, BMP4 and decreased activation of the BMP signaling pathway (pSMAD1/5/8). Accordingly, BMPs expression and BMPs-pathway activation were exacerbated in CSE KO tenocytes compared to WT tenocytes. The protective role of CSE-H<sub>2</sub>S was confirmed <i>in vivo.</i> Indeed, in aged mice, micro computed tomography revealed exacerbated Achilles' tendon calcification in CSE KO mice compared to WT. Interestingly, CSE deficiency led to reduced biomechanical strength, as the dynamic Young's modulus was significantly decreased upon increased tendon displacement. Furthermore, using a tenotomy model of CT, we found an inverse correlation between CSE expression and calcification in tendons. This was confirmed in human tendons from CT patients, which exhibited decreased CSE expression where calcification was present. In parallel experiments, we found that calcification in tenocytes was significantly reduced by addition of BAPN, a pan-inhibitor of lysyl oxidases (LOX(L)) enzymes. LOX(L) family includes 5 enzymes LOX and LOX1-4, catalysing elastin and collagen cross-links. We next investigated if the anti-calcfiying effect of H<sub>2</sub>S in CT could be mediated by inhibition of LOX(L). Indeed, in CM-stimulated tenocytes we found that H<sub>2</sub>S impacts both LOX/LOX2 expression (as CSE deficiency increased significantly LOX/LOX2 gene expression) and LOX(L) activity (as CSE deficiency increased LOX(L) activity and conversely H<sub>2</sub>S dose-dependently inhibited LOX(L) activity in WT tenocytes. Finally, <i>in vivo</i> we discovered that, CSE was inversely correlated with calcification and LOX and LOXL2 expression in mouse and human tendons. <h3>Conclusion</h3> Altogether, our results suggest that increasing H<sub>2</sub>S levels in tenocytes could represent a future strategy to prevent or decrease calcification in CT, likely via down-modulation of LOX(L) expression and activity. <h3>References</h3> [1]Castelblanco, M., et al., <i>The role of the gasotransmitter hydrogen sulfide in pathological calcification.</i> Br J Pharmacol, 2020. <b>177</b>(4): p. 778-792. [2]Kronenberg, D., et al., <i>Increased Collagen Turnover Impairs Tendon Microstructure and Stability in Integrin alpha2beta1-Deficient Mice.</i> Int J Mol Sci, 2020. <b>21</b>(8). <h3>Acknowledgements:</h3> NIL. <h3>Disclosure of Interests</h3> None Declared.